Real-time deformability cytometry: on-the-fly cell mechanical phenotyping.

We introduce real-time deformability cytometry (RT-DC) for continuous cell mechanical characterization of large populations (>100,000 cells) with analysis rates greater than 100 cells/s. RT-DC is sensitive to cytoskeletal alterations and can distinguish cell-cycle phases, track stem cell differentiation into distinct lineages and identify cell populations in whole blood by their mechanical fingerprints. This technique adds a new marker-free dimension to flow cytometry with diverse applications in biology, biotechnology and medicine.

Camera-based three-dimensional real-time particle tracking at kHz rates and Ångström accuracy.

Optical and magnetic tweezers are widely employed to probe the mechanics and activity of individual biomolecular complexes. They rely on micrometre-sized particles to detect molecular conformational changes from the particle position. Real-time particle tracking with Ångström accuracy has so far been only achieved using laser detection through photodiodes.

Fork sensing and strand switching control antagonistic activities of RecQ helicases.

RecQ helicases have essential roles in maintaining genome stability during replication and in controlling double-strand break repair by homologous recombination. Little is known about how the different RecQ helicases found in higher eukaryotes achieve their specialized and partially opposing functions. Here, we investigate the DNA unwinding of RecQ helicases from Arabidopsis thaliana, AtRECQ2 and AtRECQ3 at the single-molecule level using magnetic tweezers.

Nuclease activity of Saccharomyces cerevisiae Dna2 inhibits its potent DNA helicase activity.

Dna2 is a nuclease-helicase involved in several key pathways of eukaryotic DNA metabolism. The potent nuclease activity of Saccharomyces cerevisiae Dna2 was reported to be required for all its in vivo functions tested to date. In contrast, its helicase activity was shown to be weak, and its inactivation affected only a subset of Dna2 functions.

Efficient preparation of internally modified single-molecule constructs using nicking enzymes.

Investigations of enzymes involved in DNA metabolism have strongly benefited from the establishment of single molecule techniques. These experiments frequently require elaborate DNA substrates, which carry chemical labels or nucleic acid tertiary structures. Preparing such constructs often represents a technical challenge: long modified DNA molecules are usually produced via multi-step processes, involving low efficiency intermolecular ligations of several fragments.

Energetics at the DNA supercoiling transition.

Twisting a DNA molecule held under constant tension is accompanied by a transition from a linear to a plectonemic DNA configuration, in which part of the applied twist is absorbed in a superhelical structure. Recent experiments revealed the occurrence of an abrupt extension change at the onset of this transition. To elucidate its origin we study this abrupt DNA shortening using magnetic tweezers.

Torsional stiffness of single superparamagnetic microspheres in an external magnetic field.

We used magnetic tweezers to measure the torsional stiffness of single micrometer-sized superparamagnetic spheres as a function of the applied magnetic field. By investigating the axial fluctuations of DNA-bound microspheres, we found that considerable rotational microsphere fluctuations can occur. Quantitative noise analysis allowed us to determine the rotational stiffness of individual microspheres, which was found to saturate at high magnetic fields.